(?hem-Blol Interacttons Elsevier Pubhshlng Company, Amsterdam Printed in The Netherlands

363

T H E ESTABLISHMENT OF C LONED CELL LINES F R O M Y O S H I D A SARCOMAS H A V I N G D I F F E R E N T I A L SENSITIVITIES TO M E T H Y L E N E D I M E T H A N E S U L P H O N A T E I N V I V O A N D T H E I R CROSS- SENSITIVITY TO X-RAYS, UV A N D O T H E R A L K Y L A T I N G AGENTS

MARGARET FOX AND B W FOX

Paterson Laboratortes, Chrtstte Hospttal and Holt Radtum Instttute, Manchester (Great Brltam)

(Received September 17th, 1971) (Accepted October 1 lth, 1971)

SUMMARY

Cloned cell hnes have been estabhshed from Yoshlda ascltes tumour lines known to be sensitive and resistant to methylene d~methanesulphonate (MDMS) m vtvo The two lines have similar population doubling times and cell cycle times, but the relative lengths of different phases of the cycle differ, the S phase in particular being consider- ably longer in the resistant line Cell survwal studies by agar cloning and by the back- extrapolation method have shown that the hne sensitive to MDMS is cross-sensitive to HN2 and to UV irradiation The two cell hnes show similar sensitivities to methyl methanesulponate (MMS) and X-rays

INTRODUCTION

The Yoshlda rat sarcoma is extremely sensitive to a number of alkylatlng agents, however, resistance to such agents develops easily and some lines have been reported to be as much as 10 000 times less sensitive to the agent than the original tumour 1,2. Many attempts have been made to determine the molecular mechanisms revolved m development of resistance to alkylatlng agents, but so far these have met with little success when m wvo systems have been used BALL et a l 3 compared growth rate, chromosome number, nucleic acid, protein and free thlol content In a Yoshlda tumour sensltwe to Melphalan and one showing 100 times greater resistance A small increase in glutathlone content of the resistant tumour was demonstrated, but no other dif- ferences could be detected In a further study on the same parr of tumours, BALL and

Abbreviations MDMS, methylene dlmethanesulphonate, MMS, methyl methanesulphonate, [aH]TdR, [aH]thymldlne, YMDR, methylene dlmethanesulphonate resistant, YMoS, methylene dImethanesulphonate sensitive

Chem -Blol Interactions, 4 (1971/72) 363-375

364 MARGARET FOX, B W FOX

co-workers 4 investigated the cell kinetics and incorporation of [3H]thymldine ([3H]TdR) into D N A at various times after treatment in the two tumours Little difference in the behavlour of the two tumours could be detected except for a slowing of D N A synthesis in the cycle following treatment in the resistant line which the authors suggest could be due to repair This possibility was investigated further by BALL AND ROBERTS 5, but no &fferences in the amount of repair replication after sulphur mustard treatment of the two lines could be demonstrated

Recently GOLDENBURG et a l 6 have reported evidence suggesting that resistance to HN2 in LS178Y lymphoblasts is multlfactorlal, one factor being decreased uptake of H N 2 in resistant cells Uptake and binding of HN2 to whole cells and cellular constituents has recently been studied in Yoshlda sarcoma cells during their acquisi- tion of resistance to the drug 7 With increase in resistance, the amount of drug bound to macromolecules was found to decrease and it was concluded that changes in cell membrane transport systems played an important role m the acquisition of resistance

to HN2 The development of resistance to the alkylating agent methylene dlmethane-

sulphonate (MDMS) has been described a Normally the Yoshlda turnout is extremely sensitive to this agent, but resistance develops as a single-step process m vtvo, and is stable at a high level over considerable periods of time

Selection of Yoshlda cell lines m vitro, resistant to nitrogen mustard, has been reported by SAKURAI 9, and clones of LS178Y lymphoblasts, resistant to HN2, have been described by GOLDENBURG 1° In both the above cases, selection of resistant lines required repeated exposure to the drug

Short-term cultures of Yostuda sarcoma cells were described by SAKURAI 9, who demonstrated that after culture for 14 days in i'ttro the cells were still capable of inducing tumours in rats

Short-term culture of Yoshlda ascltes cells was also described by BALL AND ROBERTS 5 Logarithmic growth of cells derived from sulphur mustard sensitive and resistant lines could, however, be maintained only over a period of 5-10 days, and therefore accurate determinations of m vitro sensitlwties to cytotoxlc drugs were not possible even by the back-extrapolation technique and certainly not by single cell survival experiments

Culture of Yoshida tumour cells m vttro has also been described by KATSUTA et a l ~ and by GOTO AND SATO 12, and colony formation in agar using a 1 ~ basal layer and 0 5 ~ seed layer was reported ~3

In order to facilitate the study of mechanisms of resistance to and mode of action of MDMS, we have developed cloned cell lines f rom the originally sensitive Yoshida tumour and a tumour which acquired resistance to MDMS after a single exposure to the drug m vtvo

MATERIALS AND METHODS

The tumour was normally transplanted routinely into 10-week-old female Wistar rats by implanting 5-mm a pieces o f tumour tissue subcutaneously The develop-

Chem -Btol InteractiOns, 4 (1971/72) 363-375

CROSS-SENSITIVITY OF CLONED YOSHIDA TUMOUR CELLS 365

ment of lines resistant to MDMS has been described 8 Before attempts were made to

culture the sensitive and resistant hnes m vttro, the tumour was converted to an ascltes form and transplanted by mtraperltoneal mject~on of 106 ascltes cells every 7 days

Development oJ cell hnes Ascltes tumour cells were aspirated from anaesthetlsed rats 4 days after Injec-

tion of 106 Yoshlda turnout cells from either the sensmve or the reszstant cell hnes The ascltes fluid was centrifuged and the supernatant serum removed The cell pellet which contained considerable numbers of red cells was then washed twice with sahne

and once with Flscher's medium Cells were then incubated in Flscher's medium q- antibiotics and 20 Y/o horse serum for 24 h at a concentration of 106 cells/ml After the overmght incubation, large numbers of healthy looking tumour cells were visible

m both cultures The cultures were therefore harvested and centrifuged twice an medxum containing 20 % horse serum The red cells tended to sediment to the bottom of the centrifuge tube and after 2 consecutive centrlfugatlons most of the red cells were removed The tumour cells were then resuspended at a concentration of 2 10S/ml and incubated for a further 24 h During this time the cell number doubled once, cultures were then diluted 5-fold and thereafter 10-fold every 2-3 days

Clonmg procedures A method for the culture of P388 cells in medium sohdlfied with 0 35 % agar

has been described 14 Neither sensltwe nor resistant Yosh~da tumour cells attached to glass, therefore, this method was used to isolate clones

Determmatlon of cell survival Two methods were used Firstly, back-extrapolation of growth curves Th~s

method has been used previously by Fox is to determine surviving fractions m P388 cells, and its rationale has been discussed by ALEXANDER AND MIKULSK116 The rela- tionship between this method and colony survwal after plating in agar has been dis- cussed by NIAS AND FOX 14 Secondly, colony survival after plating of cells m agar was used 5000 cells were plated m 7 5 ml of medmm m 5-cm plastic petn dishes and cultures were incubated for 8-10 days before scoring the unstamed dishes under a

dissecting microscope Colonies of 200 cells or more were considered to be survivors Colomes were counted using a dissecting nucroscope, routine cell counts were made using a haemocytometer

Chemicals Nitrogen mustard was obtained from Boots Pure Drug Co and dissolved in

sterile sahne immediately before addmon to the cell cultures MMS was obtained from Eastman Kodak Ltd and MDMS was syntheslsed in this laboratory 8 All drugs were stenhsed by filtration (Mllhpore 0 22/z) before addition to the cell cultures Cells were exposed to MMS and HN2 for 60 nun before wastung twice in sahne and dilution for plating Exposure to MDMS was for 15 mm only

Chem -Btol Interacttons, 4 (1971/72) 363-375

366 MARGARET FOX, B W FOX

Irradtatton wtth X-rays and UV Cells were ~rradmted with X-rays in 16 × 125 m m screw-cap culture tubes in

the presence of medmm -520% horse serum at a dose rate of ~200 rad/nun from a Siemens 300-KeV X-ray machme

The UV source was a low-pressure gernucxdal lamp wtth a chief emitted wave- length (more than 90%) of 254 nm (Osram HNS 12) Before lrradmtlon, the cells were harvested from medmm containing serum by centnfugatlon and washed twice m sterde 0 9 % sahne They were resuspended to a concentration of 100 000 cells/ml and 1 5 ml of this suspension were p~petted into 2-cm plastic petrl dishes The cell suspension was st~rred dunng lrradmt~on using a magnetic stirrer The cell suspen- sion m sahne covered the petn dish to a depth of ,,,2 mm and the absorbance of the solution at 254 nm was 0 074, z e transnusslon was 94 % At a distance of 40 cm from the UV source, the dose rate, as determined by chemical actmometry, was l0 3 erg/mm2/sec After lrradmtlon, cells were resuspended m medium containing 20 % horse serum and dduted for determination of surwval either by plating in agar or by the back-extrapolation method

A utoradtography For deternunat~on of percentage of labelled cells, cultures were treated as fol-

lows Cultures of both cell hnes an exponentml growth 2 104--4 104 cells/ml were incubated in the presence of [3H]TdR (0 1 pCl/ml, 26 0 C1/mM) for 8 h Samples were taken at hourly intervals, the cell pellet obtained by centnfuganon (800 rev /nun for 15 mm) was washed twice with 0 9 % sahne, subjected to hypertonlc treatment with 1% sodmm otrate, then fixed and stained Autoradlographs were prepared as described prevaously 17 Shdes were exposed for 7 days, then the percentage of labelled cells determined by scoring 500 cells for each tame point for each cell lane

The procedure for deternunatlon of labelled nutoses curves was as follows Rephcate cultures of both cell hnes were pulse-labelled for 30 nun w~th [aH]TdR (0 2/zCl/ml), then washed once with Flscher's medmm supplemented wath 2/zg/ml unlabelled thynudlne Cultures were then resuspended in medium containing 2/zg/ml unlabelled thynudme Samples were subsequently taken every hour and prepared for autoradlography as described above After exposure for 7 days, shdes were scored for labelled mitoses 50 mitoses were scored for each cell hne at each time point and the fraction of labelled mitoses determined The results obtained were subjected to analysis by a computer program, by Dr C W GILBERT 18

RESULTS

Growth in hqutd medium from prlmary explants The growth rates of the cell hnes estabhshed from MDMS-sensltlve (YMDS)

and MDMS-reslstant (YMDR) tumours were measured by daily cell counts During the first 2 days after explantatlon, there was httle ewdence of increase m populataon size, however, by 4 days exponenttal growth had been estabhshed and both popula- taons grew with a sanular doublang time of 29 h After 10-15 days an culture the

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C R O S S - S E N S I T I V I T Y O F C L O N E D Y O S H I D A T U M O U R C E L L S 367

doubhng time had decreased to 18 h and by 20-25 days to 14-16 h Subsequently

the doubhng time stablhsed to between 13 and 14 h in both YMDS and YMDR cell

hnes Saturat ion density of cultures under these condit ions was similar in both cell

hnes, t e 5 105-6 105 cells/ml

Neither cell line attached to the glass surface, both grew in suspension in small

aggregates which in the sensitive cell hne were quite difficult to break up by plpet tmg

G r o w t h m s e m t - s o h d agar r n e d m m

The results of plat ing YMDS and YMDR hnes m different agar concentrat ions

is shown m Table I Concentra t ions greater than 0 25 % were lnhlb~tory to the growth

of both cell hnes At 0 2 % agar, the plat ing efficiency of the YMDS hne was initially

,,~ 5 % and very few colonies were observed m YMDR cultures Clones were isolated

f rom these cultures and after a further period of growth ( ,-~ 2 months), their p la tmg

efficiency in agar was agam tested (Table II) The plat ing efficiency of YMDS cul-

tures was on tlus occasion ~ 2 0 % and has remained at this level or slightly higher

since Again, however, very few colonies were observed after pla t ing YMDR cells,

a l though considerable growth of cells was evident within the agar and therefore the

concentra t ions used were no t lnhlbl tory We have now repeated this experiment

TABLE I

EFFECT OF DIFFERENT AGAR CONCENTRATIONS ON THE PLATING EFFICIENCY OF YOSHIDA CELL LINES

Cells were plated In 5-cm dishes and dishes were incubated for 10 days before colonies were counted using a binocular dissecting microscope

Cell hne Number o f Agar concn Number o f colonws, cells plated (%) mean 5 plates

YMoS 2000 0 2 109 -4- 6 2 0 25 10 -t- 1 5 03 2 4-05 04

YMoR 2000 0 2 5 7 4- 1 2 025 a 0 30 0 40

a Indicates considerable growth of cells within the agar, m addition to the few colonies produced

TABLE II

EFFECT OF INOCULUM SIZE ON PLATING EFFICIENCY IN TWO YOSHIDA CELL LINES

Plates were incubated for 10 days before scoring

Cell hne Number o f cells Number o f colomes, P E (%) inoculated mean 5 plates

YMDS 2000 464 4- 70 23 2 5000 898 4- 4 2 180

10000 1695 4- 6 1 16 95 YMDR 2000 43 7 -4- 6 0 2 2 a

5000 232 ± 121 46 a 10000 437 -4- 14 2 4 3 a

a Indicates considerable growth of cells within the agar, in addmon to the few colonies produced

Chem -Btol Interactwns, 4 (1971/72) 363-375

368 M A R G A R E T FOX, B W FOX

several times, each time isolating colonies from the resistant hne which do prohferate m agar, but have not been able to ~solate a hne which forms colomes with the same efficiency as do the YMvS cells The plating efficiency of both cell hnes was indepen- dent of cell moculum

Dose-response studies

Imtlal dose-response stu&es were made using the back-extrapolahon method of ALEXANDER AND MIKULSK116 The response of the YMDS hne was always confirmed by determination of single cell surwval by plating in agar, no difference m response was detectable prowdmg that the rmnlmum colony size scored exceeded 200 cells The results from both methods were pooled for the YMoS hne The back-extra- polation method only has been used for the YMDS line

The response of the two cell hnes to the original selective agent MDMS is shown m Fig 1 In a single-step change m vzvo, therefore, a 10-fold &fference m Do value was observed

z I0" _o I- U

~ 10-2 Q

10-3

o RESISTANT ~ ~ " SENSITIVE

(9 IO

°

I0: _>

I0 3

®

° ~ RESISTANT

SENSITIVE

11 0J2 0'3 DOSE of HN 2 pg/ml

,'s 2'o 3'o 3's ,'o ,'s DOSE of MDMS ~.,g/rnl

Fig 1 Sensmvlty of the two cell hnes to single doses of M D M S Cells were exposed to the drug for 15 mm then dduted and survwal measured by growth as colomes m agar or by the back-e×trapolaUon method Results from both methods for YMDS cells have been pooled to gwe the dose-surwval curve shown Results for YMDR cells are from back-extrapolaUon only Results from 3 experiments

Fig 2 Sensitivity of YMDS and YMDR cells to mtrogen mustard (HN2) Cells were exposed for 60 mm before dilution and plating for cell survival Results are mean of 3 experiments for each cell hne

The YMDR hne was also cross-resistant to HN2, for which the dose-response curves of YMDS and Y M v R are shown in Fig 2 There was a 5-fold &fference m Do values, that for the YMDS hne was 0 009 /zg/ml and for the Y M v R hne was 0 055/zg/ml The sensitivity of the two hnes to UV Irradiation also differed consider-

Chem -Bzol Interacttons, 4 (1971/72) 363-375

CROSS-SENSITIVITY OF CLONED YOSHIDA TUMOUR CELLS 369

ably The dose-response curves are shown in Fig 3 The D o value for the YMDR hne was 52 5 erg/mm 2 and that of the YMDS line was 13 5 erg/mm 2 The extrapola- tion numbers of the two lines YMDS and YMoR were 1 0 and 2 0, respectively The Do values for the two lines therefore differed by a factor o f 4

F- Io-i U

~ 1 0 - 1

10-3 [

RE

~'o ,~o ,~o 2~o 2;0 UV DOSE erg/rnm 2

o RESISTANT I ~ ° SENSITIV~

I,L

~lO -Z

I I I I j

100 200 300 400 500 DOSE(RAD)

Fig 3 Response of YMDS and YMDR cells to UV lrradmtlon Cells were exposed to UV in PO4- buffered saline, then resuspended m full growth medmm before dilution and plating Results are mean of 2 experiments for each cell hne

Fig 4 SenslUvlty of YMDS and YMvR cells to X-rays Cells were irradiated m full growth medmm at a concentration of 5 104/ml then diluted appropriately before plating m agar or for determlna- uon of growth curves Results of 2 experiments for each cell line

The sensitivities of the two cell lines to X-rays and MMS were identical and are shown in Figs 4 and 5 The Do value for X-rays was 70 rad, t e similar to that reported for P388 mouse lymphoma lines in v i tro ~9 Sensitivity to MMS, at exposure time 1 h, was also similar to that reported for P388 cells tn vl tro 2°, I e both YMDS and YMDR cells are relatively sensitive to X-rays and MMS m vl tro

C e l l cyc le p a r a m e t e r s

In exponential growth the doubling times of the two cell lines were very similar and vaned between 12 and 14 h, depending on the batch of serum used

The rate of increase of labelled cells in the two cell lines is shown in Fig 6 The YMDR line showed a consistently higher labelling index than did the YMDS line, suggesting that a greater proportion of cells are in S m the former case

Labelled mitoses curves for the two cell lines (Fig 7) confirmed this observation not only did the relative proportions of cells in S differ but also the relative lengths of the G1 and G2 phases of the cell cycle The total cell cycle ttme in the YMDR line was 12 2 h and in the YMDS line 11 5 h The durations of the vanous phases of the cell cycle were as shown in Table I I I The YMDS line showed a tugher proportion of cells out of cycle (7-9 %) than the YMDR line (2 5 ~o), which would account for the

Chem -Btol Interactions, 4 (1971/72) 363-375

370 MARGARET FOX, B W FOX

IO- Z 0 F- ( J ,v-

t~ 10-2 z

10"3

o RESISTANT • SENSITIVE ® 10o

9o

80

~ 7o

..1 61

~ 40

10 3o

211

®

s " o RESISTANT • SENSITIVE

i i I i

' 2 3 4 5 6 ' B - ' 9 TIME after the ADDITION oF[3H]TdR

0 I00 150 200 DOSE oF MMS tt~/ml

Fig 5 Dose-response curves for Yoshida cell lines exposed for 1 h to increasing doses of MMS Cells were washed twice with saline after exposure to the drug then dduted into drug-free medium, and survzval determined either be plating m agar or by back-extrapolation of growth curves

Fig 6 Rate of increase of labelled cells in the presence of 0 1 ,uC1/ml of [aH]TdR In Yoshlda cell hnes Samples were taken from cultures at hourly intervals and fixed and stained for autoradlog- raphy Slides were exposed for 7 days and percentage labelled cells determined by counting 500 cells for each point for each cell hne

FRACTION of Q LABELLED MITOSES

O9 ~ - o , ~ VOSHIDA RESISTANT

o • o

O5

O3

O 5 10 15 20 25 I t l I I I I

O ~, / ~ o YO~HIDA SENSITIVE

05

03

O t ~ 5 10 15 20 25 I 0 " o ~ I I I I

after ~H]TdR 'pulse'

Fig 7 Labelled m~toses curves for YMoS and YMDR cells pulse-labelled with 0 1 #Cz/ml [aH]TdR for 30 mzn then resuspended in normal growth medmm supplemented with unlabelled thymldlne (2 0 pg/ml)

Chem -Bzol Interactions, 4 (1971/72) 363-375

CROSS-SENSITIVITY OF C L O N E D YOSHIDA T U M O U R CELLS 371

slmdarlty of doubling rimes with different cell cycle times The doubling times of the two lines shown in Table I I I were determined in the same experiments as the labelled mitosis curves by rephcate cell counts

DISCUSSION

Cloned cultured cell lines have been established from the Yoshlda tumours which were shown to be sensitive and resistant to MDMS m v t v o 8 This differential sensitivity was retained by the hnes developed m v i t r o both by the total population and by clones derived from the original cultures, the hnes have now been cultured m v t t r o for almost 9 months with no significant change in their relative sensitivities, and the population doubling times and cell cycle times have also remained stable

In previous attempts to develop cell lines from Yoshlda ascltes tumours a variety of different media have been used, ranging from the chemically undefined ones reported by SAKURAI 9 to Eagles MEM (rmnlmal essential medium) as reported by BALL AND ROBERTS 5

In the present study, Flscher's medium was used This contains high levels of fohc acid and asparaglne which have been shown to be essential for the continued growth and high platmg effioency of L5178Y and P815Y mouse lymphoma cells m

v i t r o 21 and sirmlarly for P388 and L1210 lines (Fox, unpublished data) L-Asparaglne has also been shown to be necessary for the continued reproduction of Walker carcmo- sarcoma m v i t r o by NEUMAN AND M c C o Y 22 The ease with which cell lines were estabhshed from Yosh~da tumours, together with their relatively rapid doubhng times

• and good plating efficiency, suggests that Yoshlda turnout cells have similar nutritional reqmrements

Many different assay methods, both m v t v o and m v t t r o , have been used in the comparison of sensitivity m two cell lines, and it is therefore to be expected that there will be a wide variation in the degree of resistance reported

S A K U R A I 9 compared the minimum doses required to produce a morphological change in stained cell preparations which he termed MEC and the 50 ~ inhibition concentration IC50, t e the dose required to reduce cell growth to 5 0 ~ of control by 48 h The MEC and IC50 for nitrogen mustard were found to differ by a factor of 10, the latter being lower The dose required to reduce the number of tumour takes to 5 0 ~ of control after treatment of a standard number of cells m v t t r o was compared by BALL e t a l a with the rate of increase of tumour weight after treatment of bilaterally transplanted sensitive and resistant tumours Fox s compared the rate of mcrease of tumour volume after treatment, whilst in a more recent paper BALL AND ROBERTS 5 compared the dose to give 10 ~ survival in their two cell lines and quoted a resistance index

In the present study we have been able to determine dose-response curves either for survival of colony-forn~ng abdlty or for growth of surviving cells at the same rate as the control population, and as a result a number of different parameters can be compared, e g comparison of cell survival in the two hnes after the same dose of HN2 results in a 100-fold difference Jn sensmvity, whereas comparison of the Do

C h e m -Bto l In terac t ions , 4 (1971/72) 363-375

372 MARGARET FOX, B W FOX

slopes shows only a 6-fold difference In the case of MDMS, if a dose of 15/~g/ml is considered, then the two hnes show a 10 000-fold difference in sensitivity, whereas a comparison of the Do values results m much smaller d~fferences

In terms of m vtvo and m vttro assays, it ~s interesting to note that a dose of 15/tg/ml would reduce the sensitive population to H1 In 10 -4 and that the eqmv-

alent dose, t e 15 mg/kg, cured all animals beanng the senslnve tumour when injected 6 days after tumour implantation a

D~fferences m sensttlvlty between closely related cell hnes to X-rays, UV and

cytotomc drugs may be due to a variety of factors, many of which have been discussed by GOLDENBUP.G l° The most important of these are the following (1 ) Smce it is

well known that different phases of the mitotic cycle of several cell hnes are differen- tially sensitive to X-rays 2a and cytotoxlc drugs2% then cell hnes showing differences in the relanve proportions of cells at various stages of the cycle may show different sensitivities, (2 ) differences in the relatwe uptake or rate of metabohsm of cytotoxlc agents, (3 ) differences in repair capaoty, (4 ) increased cellular concentration of protecnve agents (th~ol groups) or a comblnanon of any number of these factors

It is well known that the dose-response curve of an asynchronous population, at least on the exponential part of the curve, ~s largely determined by the most resistant fraction 2s HeLa cells have been shown to be more resistant to MMS in the S phase of the cycle 26, as have Chinese hamster HA 1 cells 24 In addition, sensltwlty to MMS

has been shown to be greater in a subhne of HeLa cells which had an elongated G1 phase 26 A subhne of V79 cells with an elongated G~ has also been shown to be more sensitive to X-rays than the parent line 27

In the Yosh~da cultures studied here, the S phase, presumably the more resistant phase to both X-rays and MMS, is almost twice as long m the YMDR hne as in the YMDS hne (Fig 3 and Table III and, the G~ phases constitute 27 0 and 31 2 % of the

T A B L E I I I

DURATION OF THE CELL CYCLE AND STAGES OF THE CELL CYCLE IN THE TWO YOSHIDA CELL LINES

Va lues g iven a r e de r ived f r o m the d a t a s h o w n in F ig 7 by the m e t h o d s desc r ibed m r e f 13

Cell hne Length of cell cycle phase (h)

T c G 1 G 2 S T D

YMDR 12 25 -4- 0 19 3 31 4- 0 22 1 36 ! 007 7 58 ± 0 16 13-14 YMDS 1151 dz027 3644-034 2904-009 4974-020 13-14

total cycle time in the respective cell hnes These differences in proportions of cells at various stages of the cycle do not, however, result m a d~fference in overall sensitiv- ity of the asynchronous population to either X-rays or MMS

S has been reported to be the phase most resistant to nitrogen mustard and to sulphur mustard m HeLa and Chinese hamster cells 24 and mouse L cells 28,29 The

differences m sensitivity between the two Yoshlda hnes could therefore possibly be explained on th~s basis alone However, the cell hne resistant to nitrogen mustard also shows cross-resistance to UV Chinese hamster cells have been shown to be most resistant to cell kllhng by UV in G1 and G2 and most senslnve m S (ref 30), and HeLa cells also show a slmdar d~strlbutlon of phase sensltwlty 31 Therefore, if cell

Chem -Blol Interacttons, 4 (1971/72) 363-375

CROSS-SENSITIVITY OF CLONED YOSHIDA TUMOUR CELLS 373

age distribution is determining overall sensitivity of the asynchronous population, one

would expect the YMoR hne to be sensitive to UV One must thus conclude that the differences in sensitivity between the two Yoshlda cell lines to UV, HN2 and MDMS are determined by factors other than cell age distributions

The possibility exists in the case of MDMS and HN 2 that resistance is due to the ability of the resistant line either to exclude the drug or to metabohse it more quickly A reduction in the rate of uptake of HN2 in resistant L5178Y lymphoblasts has been reported by GOLDENBURG et al 6 and in resistant Yoshlda sarcoma cells by INABA AND SAKURAI 7 In the latter case, differences in sensitivity ranged from 100 to 1000-fold, but the binding of the drug to DNA, RNA and protein was found to differ

by a factor of 6 at most between sensitive and resistant lines In the Yoshida sarcoma cell lines described here, resistance to HN2 was found

to be linked to resistance to UV light There must therefore be a significant contribu- tion of factors other than reduced drug uptake to the observed difference in sensitivity

In bacteria, sensitivity to UV and to HN2 tends to be correlated with one group of loci a2, whereas sensitivity to X-rays and MMS is correlated with another 33 In

the yeast Saccharomyces cerevtstae also, X-ray sensitivity could be correlated with MMS sensitivity whilst sensitivity to HN2 was correlated with sensitivity to UV (ref 34) HN 2 and UV have been shown to induce cross-links in DNA a5 36, whereas

X-rays and MMS are known only to induce single-strand breaks in both bacterio- phage and mammalian cells 37-4° It seems likely therefore that in the Yoshlda system

described here, some components of HN 2 damage are repaired in the same way as UV and MDMS damage and that conversely there are components of X-ray damage which are repaired in the same way as MMS damage These findings in the Yoshida system also agree with the observations of REITER AND STRAUSS 41 who demonstrated that a (uvr-) strain of B subtths showed no cross-sensitivity to MMS

In other mammalian cell lines in vttro the correlations so far achieved have been less clear TODD AND HELLEWELL 42 reported that a slow-growing line of Chinese hamster ceils (V79-79) which was X-ray sensitive was also more sensitive to UV irra- diation, and the M4 clone isolated by Fox AND NIAS 43 and characterlsed as having an elongated G1 (ref 26) has been shown to be also more sensitive to UV 44 and to X-rays (NIAS AND FOX, unpublished)

It has been suggested by BALL AND ROBERTS 5, after comparison of levels of

[aSS]mustard binding in a number of cell lines, that their YS cells lack some step in the repair process which occurred in YR cells and in other cell lines, e g HeLa and Chinese hamster V79 The difference was, however, shown not to lie in the excision- repair step, as measured by CsC1 density gradient centrifugatlon, and rate of loss of alkyl groups from labelled DNA These authors suggested that recombination repair may be deficient in their YS cells, and the fact that the cell line described here is not only sensitive to HN 2 and MDMS but also to UV would tend to support this idea RuaP et al 4s claim to have demonstrated the existence of recombination repair in mouse LS178Y cells

It is therefore proposed that In addition to the possible differences in the rate of uptake and binding of the drug to cellular constituents which may partially explain

Chem -Blol Interacttons, 4 (1971/72) 363-375

374 MARGARET FOX, B W FOX

th e d i f f e rences in sens i t iv i ty , t he d a t a p r e s e n t e d h e r e s u p p o r t t he i de a t h a t t he Y M D S

cells a re def ic ien t m s o m e t y p e o f r e p a i r m e c h a n i s m w h i c h is p r e s e n t in Y M D R cells

a n d t h a t t h i s m e c h a n i s m is n e c e s s a r y f o r t he r e p a i r o f a t ype o f d a m a g e c o m m o n to

U V , H N 2 a n d M D M S T h e s t ab i l i t y o f t he t w o cell l ines a n d t he r e l a t ive ly l a rge

d i f f e rences in s ens i t i v i t y s h o u l d f a c l h t a t e t he f u r t h e r i n v e s t i g a t i o n o f t h i s m e c h a n i s m

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